U.S. patent number 5,656,275 [Application Number 08/480,882] was granted by the patent office on 1997-08-12 for recombinant raccoon pox virus comprising the dna encoding the nucleocapsid protein of feline infectious peritonitis virus.
This patent grant is currently assigned to American Home Products Corporation. Invention is credited to Lloyd Chavez, Hsien-Jue Chu, Terri Wasmoen.
United States Patent |
5,656,275 |
Wasmoen , et al. |
August 12, 1997 |
Recombinant raccoon pox virus comprising the DNA encoding the
nucleocapsid protein of feline infectious peritonitis virus
Abstract
This invention provides a recombinant raccoon poxvirus that
expresses the nucleocapsid and transmembrane proteins of Feline
Infectious Peritionitis Virus. The recombinant viruses are useful
as vaccines, either alone or in combination with carriers and
adjuvants.
Inventors: |
Wasmoen; Terri (Fort Dodge,
IA), Chavez; Lloyd (Fort Dodge, IA), Chu; Hsien-Jue
(Fort Dodge, IA) |
Assignee: |
American Home Products
Corporation (Madison, NJ)
|
Family
ID: |
22420074 |
Appl.
No.: |
08/480,882 |
Filed: |
June 7, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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125516 |
Sep 22, 1993 |
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Current U.S.
Class: |
424/199.1;
424/221.1; 424/211.1; 424/208.1; 424/274.1; 435/235.1; 435/320.1;
424/263.1; 424/216.1; 424/233.1; 424/202.1; 424/201.1; 424/819;
424/232.1; 424/207.1; 424/229.1 |
Current CPC
Class: |
A61P
31/12 (20180101); C07K 14/005 (20130101); C12N
15/86 (20130101); A61K 39/00 (20130101); C12N
2770/20022 (20130101); C12N 2710/24022 (20130101); Y10S
424/819 (20130101); C12N 2710/24143 (20130101); C07K
2319/00 (20130101) |
Current International
Class: |
C12N
15/863 (20060101); C07K 14/165 (20060101); C07K
14/005 (20060101); C07K 14/065 (20060101); A61K
39/00 (20060101); A61K 039/295 (); A61K 039/275 ();
A61K 039/215 (); A61K 039/21 (); A61K 039/265 ();
A61K 039/118 (); C12N 015/00 (); C12N 007/00 ();
A61K 039/23 (); A61K 039/155 (); A61K
039/125 () |
Field of
Search: |
;424/199.1,232.1,221.1,202.1,201.1,819,207.1,233.1,211.1,119.1,232,216.1,208.1
;435/320.1,235.1 |
References Cited
[Referenced By]
U.S. Patent Documents
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5242686 |
September 1993 |
Chu et al. |
5266313 |
November 1993 |
Esposito et al. |
|
Foreign Patent Documents
Primary Examiner: Caputa; Anthony C.
Attorney, Agent or Firm: Darby & Darby
Parent Case Text
This is a continuation of application Ser. No. 08/125,516, filed
Sep. 22, 1993, now abandoned.
Claims
What is claimed is:
1. A recombinant raccoon poxvirus having at least one internal gene
comprising a DNA sequence encoding the nucleocapsid (N) protein of
Feline Infectious Peritonitis Virus (FIPV).
2. The recombinant raccoon poxvirus of claim 1 wherein said
internal gene has the amino acid sequence of SEQ ID NO: 6.
3. A vaccine comprising:
a recombinant raccoon poxvirus having at least one internal gene
comprising a DNA sequence encoding the nucleocapsid (N) protein of
Feline Infectious Peritonitis Virus (FIPV), and
a pharmaceutically acceptable carrier.
4. The vaccine of claim 3 wherein said internal gene has the amino
acid sequence of SEQ ID NO: 6.
5. The vaccine of claim 3 further comprising inactivated or
attenuated viruses selected from the group consisting of feline
leukemia virus, feline panleucopenia virus, feline rhinotracheitis
virus, feline calicivirus, feline immunodeficiency virus, feline
herpesvirus, feline enteric coronavirus, and mixtures thereof.
6. The vaccine of claim 3 further comprising organisms selected
from the group consisting of inactivated or attenuated feline
Chlamydia psittaci, Microsporum canis, and mixtures thereof.
7. The vaccine of claim 3, further comprising a pharmaceutically
acceptable adjuvant.
8. A method for preventing disease caused by Feline Infectious
Peritonitis Virus (FIPV), comprising administering to a feline in
need of such treatment via a subcutaneous or intramuscular route a
vaccine comprising a recombinant raccoon poxvirus having at least
one internal gene comprising a DNA sequence encoding the
nucleocapsid (N) protein of FIPV.
9. The method of claim 8 wherein said internal gene has the amino
acid sequence of SEQ ID NO: 6.
Description
FIELD OF THE INVENTION
The present invention pertains to the prophylaxis of disease caused
by feline infectious peritonitis virus (FIPV), using recombinant
raccoon pox viruses (RRPVs) expressing the nucleocapsid and
transmembrane proteins of FIPV as vaccines.
BACKGROUND OF THE INVENTION
Feline infectious peritonitis virus (FIPV) induces a systemic
infection in cats that is often fatal. The effusive form of the
disease is characterized by accumulation of fibrinous ascitic
fluid. The non-effusive form of the disease is characterized by
granulomatous lesions in multiple organs including, but not limited
to, liver, spleen, kidneys, lung, and intestines. Reviewed in
Barlough, J. E. and C. A. Stoddart. Feline Coronaviral Infections
in C. E. Greene (Ed.). Infectious Diseases of the Dog and Cats. W.
B. Saunders Co., Philadelphia, Pa., 1990, pp. 300-312.
Feline infectious peritonitis virus is a coronavirus composed of
three major structural proteins: The S (spike) protein, the E1 or M
(transmembrane) protein, and the N (nucleocapsid) protein. Venema
et al., Virology 181: 327-335, 1991 and Dale, et al., EPO
0,376,744.
Prior vaccines intended to prevent FIPV infection have actually
been shown to exacerbate the disease caused by this virus.
Pedersen, N. C. and J. W. Black, Am. J. Vet. Res. 44: 229-234,
1983; Vennema H., et al., J. Virol. 64: 1407-1409, 1990; Barlough,
J. E., Can. J. Comp. Med. 49: 303-307, 1985; Barlough J. E. et al.,
Lab. Anim. Sci. 34: 592-597, 1984; Stoddart, C. A., et al., Res.
Vet. Sci. 45: 383-388, 1988; and Pedersen, N. C., Adv. Vet. Sci.
Comp. Med. 33: 413-428, 1989. This phenomenon apparently reflects
an immune enhancement of infection mediated by immunoglobulins
produced in response to the virus, in particular by those
antibodies directed against the S protein. Olsen C. W. et al., J.
Virol. 4: 175-189, 1981. Therefore, the best candidate vaccine for
prophylaxis of this disease would be a preparation that induces
strong cell-mediated immunity in the absence of enhancing
antibodies. This could be accomplished with a vaccine that lacks
the outer envelope protein but contains the other structural
proteins of FIPV (N and E1). Prior attempts to vaccinate cats with
a recombinant vaccinia virus expressing the N or E1 proteins of
FIPV, however, have failed to induce strong protective immunity.
Venema et al., Virology 181:327-335, 1991 and Dale, et al.,
European Patent Application 0,376,744. See also, Venema, European
Patent Application 0,411,684.
What is needed in the art, therefore, is an effective vaccine
against FIPV that utilizes the N and E1 proteins, or segments
therefrom, as immunogens.
SUMMARY OF THE INVENTION
The present invention pertains to the induction of protective
immunity to FIPV in cats. One object of the invention is to provide
recombinant raccoon poxviruses containing the genes for the FIPV N
or M/E1 proteins (RRPV-N and RRPV-E1, respectively).
A further object of the invention is to provide a feline vaccine
comprising RRPV-N or RRPV-E1, either singly or in combination, or
in combination with other viruses, bacteria, or fungi that have
been inactivated or attenuated.
A still further object of the invention is to provide a method for
preventing disease caused by FIPV, by administering to a feline in
need of such treatment a vaccine comprising RRPV-N, RRPV-E1, or
combinations thereof.
These and other objects and advantages, which will be apparent from
this specification, are achieved by the invention described
below.
DESCRIPTION OF THE DRAWINGS
FIGS. 1A through 1E, when joined at respective match lines A--A
through D--D, illustrate the nucleotide and amino acid sequence of
the FIPV E1 protein and the (SEQ ID NO: 1) and (SEQ ID NO: 5),
respectively. (FIGS. 1A and 1B, respectively.)
FIGS. 2A through 2G, when joined at respective match lines A--A
through F--F, illustrate the nucleotide and amino acid sequence of
the FIPV N protein (SEQ ID NO: 2) and (SEQ ID NO:6),
respectively.
FIGS. 3A and 3B illustrate the plasmid used to clone the genes
encoding the FIPV E1 and N proteins.
FIGS. 4A schematically shows the pSC11 transfer plasmid used to
create RRPVs encoding the FIPV E1 protein.
FIGS. 4B schematically shows the pSC11 transfer plasmid used to
create RRPVs encoding the N protein.
FIGS. 5A to 5F, when joined at respective match lines A--A through
E--E, illustrate the nucleotide sequence of pSC11 FIPV E1.
FIGS. 6A through 6F, when joined at respective match lines A--A
through E--E, illustrate the nucleotide sequence of pSC11 FIPV
N.
FIG. 7 is a photograph of an ethidium bromide-stained agarose gel
showing the analysis of RRPV-FIPV N and RRPV-FIPV E1 by polymerase
chain reaction.
FIG. 8 is an imunoblot illustrating the detection of FIPV N and E1
proteins in virally infected cell lysates.
DETAILED DESCRIPTION OF THE INVENTION
The vaccine of the present invention may be prepared by creating
recombinant raccoon poxviruses (RRPVs) containing the genes
encoding the N or E1 proteins of FIPV or immunogenic fragments
thereof. These genes are first inserted into a transfer plasmid,
which is then introduced into appropriate host cells that have been
previously infected with a raccoon poxvirus. As a result, the DNA
from the transfer plasmid is incorporated into the poxvirus DNA by
homologous recombination, producing the RRPVs that are released
from the cells.
DNA encoding the FIPV N or E1 proteins is inserted into the
transfer plasmid immediately downstream of a poxvirus promoter. In
a preferred embodiment, the early/late 7.5 Kd protein promoter of
vaccinia virus is used; however, alternate promoter elements that
are functional in poxviruses can also be used.
The preferred transfer plasmid also contains a beta-galactosidase
marker gene, which allows for selection and detection of the
plasmid DNA sequences in recombinant viruses. It will be obvious to
one skilled in the art that alternative selectable marker genes,
such as the neomycin resistance gene or the E. coli gpt gene or
others, can be used to practice the invention. Flanking the foreign
gene of interest and the selectable marker gene are thymidine
kinase DNA sequences, which facilitate recombinatorial integration
of the plasmid DNA sequences into the raccoon poxvirus DNA.
Recombinant viruses expressing the FIPV N or E1 genes are prepared
by first infecting a susceptible cell line such as Vero (ATCC CCL
81), BSC-1 (ATCC CCL 26), RAT-2 (ATCC CRL 1764), or CRFK (ATCC CCL
941) with wild type raccoon poxvirus (ATCC VR-838 or similar
isolates, such as, for example, RCNV71-I-85A). Transfer plasmid DNA
containing the E1 or N gene is then transfected into the infected
cells using cationic liposome-mediated transfection, or other
suitable techniques such as electroporation or calcium phosphate
precipitation. Virus replication is allowed to proceed until
cytopathic effects are noted in all cells.
Incorporation of the FIPV E1 or N genes into poxvirus DNA is
accompanied by disruption of the viral thymidine kinase gene.
Therefore, virus harvested from this infection may be isolated by
selecting for the absence of a thymidine kinase gene; this is
achieved by growth on tk-RAT-2 cells (ATCC CRL 1764) in the
presence of 5-bromodeoxyuridine. Viruses containing a gene insert
from the transfer plasmid are further identified by the appearance
of a blue plaque color when grown in the presence of a chromogenic
substrate for beta-galactosidase such as X-gal.
Viral plaques that survive these selection and screening procedures
are then subjected to several cycles of plaque purification.
Subsequently, the presence of the E1 or N genes is confirmed by
polymerase chain reaction technology, and the presence of E1 or N
protein is confirmed by immunoblot analysis using specific
antibodies. These viruses are designated RRPV-FIPV E1 and RRPV-FIPV
N, respectively.
In a further embodiment of the present invention, the genes
encoding N and E1 were inserted into a single transfer plasmid.
Introduction of this plasmid into cells previously infected with
wild-type raccoon poxvirus results in the production of recombinant
viruses that express both proteins simultaneously (RRPV-FIPV
E1/N).
In a still further embodiment, RRPVs can be produced that express
less-than-full-length segments of the FIPV E and N proteins. The
techniques used to engineer transfer plasmids encoding partial
sequences of E1 and N are well-known and widely used in the art, as
are the methods for production and screening of RRPVs as detailed
in this specification. For example, introduction of
oligonucleotides containing a stop codon at various points along E1
or N DNA will produce a nested set of carboyxterminal-truncated
versions of that gene, which can then be incorporated into RRPVs.
It will be apparent to one of ordinary skill in the art that
systematic screening of such recombinant RRPVs can establish
whether the intact protein, or subfragments thereof, are most
preferred in practicing the present invention. Furthermore, as
stated above, DNA encoding different fragments of E1 and N can be
used in a combination vaccine after incorporation into the same, or
different, RRPVs.
For vaccine preparation, susceptible cells such as those listed
above are infected with RRPVs at a multiplicity Of infection (MOI)
of 0.1 infectious units/cell or less. In this specification, an
infectious unit is defined as a Tissue Culture Infectious Dose
(TCID.sub.50), an amount of virus yielding 50% infection under
defined conditions. A method for TCID.sub.50 determination is
detailed in Example 1 below. When cytopathology is noted in >90%
of the cells, the infected cells and extracellular fluids (both of
which contain viruses) are harvested as a single virus-cell
lysate.
The highly concentrated virus stock to be used as a vaccine may be
stored frozen (-50.degree. C. or colder) or lyophilized until the
time of use. Compounds such as NZ-amine, dextrose, gelatin or
others designed to stabilize the virus during freezing and
lyophilization may be added. The virus initially present in the
virus-cell lysate may be further concentrated using commercially
available equipment.
Typically, the concentration of virus in the vaccine formulation
will be a minimum of 10.sup.6.5 TCID.sub.50 per dose, but will
typically be in the range of 10.sup.7.0 to 10.sup.9.0 TCID.sub.50
per dose. At the time of vaccination, the virus is thawed (if
frozen) or, if lyophilized, is reconstituted with a
physiologically-acceptable carrier such as deionized water, saline,
phosphate buffered saline, or the like.
The present invention is not limited to native (i.e.
replication-competent) RRPVs. The virus-cell lysate can be
subjected to treatments commonly used in the art to inactivate
viruses. A composition comprising inactivated virus and expressed
protein will be effective in eliciting protective immunity against
FIPV if it contains a sufficient quantity of FIPV protein. This
type of vaccine would provide added assurance that recipient
felines will not be exposed to infectious FIPV as a consequence of
vaccination. In addition, a physiologically-acceptable adjuvant may
be added to the virus, such as EMA 31 (Ethylene Maleic anhydride
31) (Monsanto Co., St. Louis, Mo.), NEOCRYL (Polyvinyl Chemical
Industries, Wilmington, Mass.), MVP (Modern Veterinary Products,
Omaha, Nebr.), Squalene, PLURONIC L121
(polyoxypropylene-polyoxyethylene block copolymer) or the like.
Individual raccoon poxviruses expressing the N or E1 genes may be
mixed together for vaccination. Furthermore, the virus may be mixed
with additional inactivated or attenuated viruses, bacteria, or
fungi such as feline leukemia virus, feline panleukopenia virus,
feline rhinotracheitis virus, feline calicivirus, feline
immunodeficiency virus, feline herpesvirus, feline enteric
coronavirus, feline Chlamydia psittaci, Microsporum canis, or
others. In addition, antigens from the above-cited organisms may be
incorporated into combination vaccines. These antigens may be
purified from natural sources or from recombinant expression
systems, or may comprise individual subunits of the antigen or
synthetic peptides derived therefrom.
In a further embodiment of the present invention, live or
inactivated RRPV virus-cell lysates can be incorporated into
liposomes, or encapsulated in peptide-, protein-, or
polysaccharide-based microcapsules prior to administration, using
means that are known in the art.
The final vaccine is administered to cats in a volume that may
range from about 0.5 to about 5 ml. The vaccine can be administered
by subcutaneous, intramuscular, oral intradermal, or intranasal
routes. The number of injections and their temporal spacing may be
varied. One to three vaccinations administered at intervals of one
to three weeks are usually effective.
The following examples are intended to further illustrate the
invention without limiting its scope. The techniques used to infect
and transfect cells, plaque purify virus, perform immunoblot
analysis are widely practiced in the art.
EXAMPLE 1
GENERATION OF RECOMBINANT RACCOON POX VIRUSES EXPRESSING FIPV N AND
E1 GENES
1. Cloning of FIPV N and E1 Genes and Preparation of Transfer
Plasmids
The sequences of the E1 SEQ. ID. NO. 1 and N SEQ. ID. NO. 2 genes
used in the present invention are shown in FIGS. 1A to 1E and 2A to
2G, respectively, of the specification. The methods for cloning of
the N and E1 genes of FIPV and their insertion into a pSC11
transfer vector are detailed in European Patent Application
0,376,744, which is incorporated by reference. The plasmid used to
clone the cDNA for the E1 and N genes is shown in FIGS. 3A and 3B.
The pSC11 plasmids carrying the E1 and N genes are shown in FIGS.
4B and 4 B, respectively. The sequences of these plasmids are shown
in FIGS. 5A to 5F (SEQ ID NO: 3) and FIGS. 6A to 6F (SEQ ID NO:
4).
To construct a pSC11 transfer plasmid containing both N and E1
genes, a 1.0 kb DNA fragment containing the vaccinia 7.5 promoter
and the E1 gene was inserted downstream of the N gene in pSC11-FIPV
N. The resulting plasmid was designated pSC11-FIPV N/E1.
2. Preparation of Recombinant Raccoon Poxviruses (RRPVs)
Monolayers of Vero cells (ATCC CCL 81) that were 80% confluent
(approximately 5.times.10.sup.6 cells/100 mm tissue culture dish)
were infected for 30-60 minutes at 37.degree. C. with wild-type
raccoon pox virus (ATCC VR-338) at a multiplicity of infection MOI)
of 0.1 TCID.sub.50 /cell. The medium (2 ml) consisted of Eagle's
Minimum Essential Medium ("MEM", Gibco BRL #410-1500) containing
0.05% lactalbumin hydrolysate and 15 .mu.g/ml gentamicin sulfate
and adjusted to pH 7.2 with sodium bicarbonate. After infection,
the medium was removed and the cells were transfected with the
pSC11-FIPV N, pSC11-FIPV E1, or pSC11 N/E1 transfer plasmid by
cationic liposome-mediated transfection using
Dioctadecylamidoglycyclsperimine-4-trifluoroacetic acid,
(TRANSFECTAM) (Promega Corporation, Madison, Wis.) and
dioleoyloxy)propyl-N,N,N-trimethylammonium methyl sulfate (DOTAP)
(Boehringer Mannheim, Indianapolis, Ind.), respectively, per
manufacturer's instructions. The cells were incubated with the
DNA-liposomes mixture in 3 ml of MEM containing 5% fetal bovine
serum (FBS) overnight at 37.degree. C. (5% CO.sub.2), after which
the medium was replaced with 8 ml of fresh MEM-5% FBS. The
transfected cells were incubated at 37.degree. C. (5% CO.sub.2)
until greater than 80% showed cytopathic effects (CPE), which took
approximately 3-4 days. The virus-cell lysates were then removed
from the plates and subjected to two cycles of freeze-thawing
before storage at -70.degree. C.
3. Isolation of Recombinant Raccoon Pox Virus Carrying the FIPV N
Gene
RRPVs carrying the FIPV N gene (RRPV-FIPV N) were isolated and
purified from the pSC11 -FIPV N Vero cell transfection by standard
viral plaque purification methods. Monolayers of Vero cells (50-80%
confluent) were infected with 2 ml of ten-fold serial dilutions
(10.sup.-1 to 10.sup.-3) of the viral-cell lysates for 1 hour at
37.degree. C. After incubation, the media was removed and the
infected cells were overlaid with 8-10 ml of 1.25% Noble agar
containing MEM/5% FBS. The infected cells were then incubated for
3-4 days at 37.degree. C. (5% CO.sub.2), and overlaid again with 4
ml of 1.25% Nobel agar containing 0.5 X PBS and 600 .mu.g/ml
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-gal,
States Biochemical Cleveland, Ohio). The plates were incubated at
37.degree. C. (5% CO.sub.2) for 4-16 hours, until blue (i.e.
.beta.-galactosidase positive) viral plaques were observed. The
recombinant viral plaques were picked with sterile blunt needles
attached to a 1 cc syringe, suspended in 0.5 ml of 0.25 .mu.g/ml
trypsin, vortexed vigorously, and incubated at 37.degree. C. for
15-30 min. The disrupted viral plaques were then inoculated onto
5.times.10.sup.5 Vero cells in 25 cm.sup.2 flasks and incubated at
37.degree. C. (5% CO.sub.2) until greater than 80% CPE was
observed. The viral-cell lysates containing RRPV-FIPV N were
subjected to two cycles of freeze-thawing and stored at -70.degree.
C. Six individual RRPV-FIPV N clones were selected and
plaque-purified five times as described above.
Isolation of Recombinant Raccoon Pox Virus Containing the FIPV E1
Gene
RRPVs carrying the FIPV E1 gene (RRPV-FIPV E1) were isolated and
purified from pSC11-FIPV E1-transfected Vero cells using the
methods described for rRPV-FIPV N, with some modifications. In this
case, thymidine kinase deficient (tk-) RRPVs from the initial
virus-cell lysates were selected on tk-RAT-2 cells (ATCC CRL 1764).
This was performed by inoculating 1 ml of the initial virus-cell
lysate onto a monolayer of RAT-2 cells in a 75 cm.sup.2 flask
(approximately 5.times.10.sup.6 cells) in the presence of
5-bromodeoxyuridine (BrdU) at 30 .mu.g/ml in MEM. The infected
monolayer was incubated at 37.degree. C. (5% CO.sub.2) for 3-4 days
until greater than 70% CPE was observed. The tk-virus-cell lysates
were subjected to two cycles of freeze-thawing before storage at
-70.degree. C. Two individual RRPV-FIPV E1 clones were selected and
subjected to six cycles of plaque purification as described above
for RRPV-FIPV N.
5. Confirmation of FIPV N and E1 Genes in RRPV by Polymerase Chain
Reaction
The presence of the FIPV N and E1 genes in the RRPVs was confirmed
using the polymerase chain reaction (PCR). 90 .mu.l of a virus-cell
lysate were incubated with 10 .mu.l of tenfold concentrated PCR
lysis buffer (100 mM Tris-HCL buffer, pH 8.5; 500 mM KCl; 25 mM
MgCl.sub.2 ; 5% Tween 20 (polyoxyethylenesorbitan monolaurate); 3
mg/ml Proteinase K) for 16 hours at 50.degree. C., then boiled for
10 min. 10 .mu.l of this lysate was used in the PCR. PCR was
performed in 100 .mu.l of 10 mM Tris-HCL buffer, pH 8.3; 50 mM KCl;
200 uM of each deoxyribonucleotide triphosphate, 1.5 mM MgCl.sub.2
; 30 pmoles of each oligonucleotide primer; and 2.5 units of
AMPLITAQ DNA polymerase (Taq DNA polymerase) (Perkin-Elmer Cetus,
Norwalk, Conn.). The primers used in the PCR for FIPV N were:
corresponding to nucleotides 68-91 and 721-744 of the FIPV N open
reading frame (SEQ ID NO: 2) (primers 1 and 2, respectively). The
primers used in the PCR for FIPV E1 were:
corresponding to nucleotides 334-355 and 721-742 of the FIPV E1
open reading frame SEQ. ID. NO. 1 (primers 3 and 4, respectively).
The PCR amplifications were performed in a DNA Thermal Cycler
Perkin-Elmer Cetus) by first heating the reaction mixes to
94.degree. C. for denaturation, and then performing 35 cycles of
amplification, each consisting of 1 min at 95.degree. C., 1 min at
55.degree. C., 2 min at 72.degree. C., and, on the last cycle, a
final incubation of 8 min at 72.degree. C. 10 .mu.l of the PCR
products were resolved by electrophoresis in a horizontal-submarine
4% NUSIEVE (agarose) gel (FMC BioProducts, Rockland, Me.) in TAE
buffer (40 mM Tris base, 20 mM sodium acetate, 1 mM EDTA, pH 7.2)
by applying 5 V/cm for 1-2 hours. The DNA products were visualized
by staining the gels with ethidium bromide.
PCR amplifications with the FIPV N and E1 primers gave DNA
fragments of 676 and 408 nucleotides, respectively (FIG. 7). PCR
amplifications using the pSC11 FIPV N and E1 transfer plasmids
served as positive controls, and showed products of the predicted
sizes. PCR amplifications using wild-type raccoon pox virus-Vero
cell lysates served as a negative control, and no PCR products were
observed in those samples.
Confirmation of RRPV FIPV N and E1 Protein Expression by Immunoblot
Analysis
Confluent monolayers of Vero cells in a 25 cm.sup.2 flask
(1-2.times.10.sup.6 cells) were infected with clones of either
RRPV-FIPV N or RRPV-FIPV E1 at an MOI of 0.1. The infected cell
were incubated at 37.degree. C. (5% CO.sub.2) for 2-3 days until
approximately 80% of the cells showed cytopathic effects. A
virus-cell lysate was prepared, and 20 .mu.l of the sample were
added to 5 .mu.l of 5 X Laemmli sample buffer (0.3M Tris-HCI
buffer, pH 6.8, containing 5% SDS, 50% glycerol, 0.4% bromophenol
blue, and 3% 2-.beta.-mercaptoethanol) and heated at 95.degree. C.
for 5 min. The denatured protein samples were separated By
SDS/polyacrylamide electrophoresis using a 4-15% gradient
polyacrylamide gel as described previously. Maniatis et al.,
Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor
Press. After electrophoresis, the proteins were transferred to
nitrocellulose (Bio-Rad Laboratories, Hercules, Calif.) by
electrotransfer using a Bio-Rad transfer apparatus per
manufacturer's instructions. The transfer was performed in 25 mM
Tris-HCI buffer, containing 0.2M glycine and 20% methanol, for 45
minutes at 50 V with constant current.
FIPV N and E1 proteins were visualized on the nitrocellulose filter
using specific antibodies. Davis et al., Basic Methods in Molecular
Biology, 1986, Elsevier Science Publishing Company, New York, N.Y.
The filter was rinsed in phosphate buffered saline pH 7.4
containing 0.1% Tween-20 (polyoxyethylenesorbitan monolaurate)
("PBS-TW"), after which non-specific sites were blocked by
overnight incubation at 4.degree. C. in PBS containing 1% bovine
serum albumin (PBS-BSA) followed by a 15 min wash in PBS-TW. The
filter was then incubated for 30 min at room temperature with
anti-FIPV antibodies, which consisted of ascites fluid from a FIPV
(strain 79-1146)-infected cat, diluted 1:100 in PBS-TW containing
1% BSA ("PBS-TW-BSA"). After four 5 min washes in PBS-TW, the
filter was incubated for 30 min at room temperature with a
secondary antibody consisting of biotin-labeled mouse anti-cat IgG
antibody (Kirkegaard & Perry Laboratories Inc., Gaithersburg,
Md.) that had been diluted 1:2000 in PBS-TW-BSA, followed by four 5
min washes in PBS-TW. The filter was then incubated for 30 min at
room temperature with horseradish peroxidase-conjugated
streptavidin (Kirkegaard a Perry Laboratories Inc.) that had been
diluted 1:1000 in PBS-TW. After the filter was washed four times (5
min each) in PBS-TW, the antigen-antibody complexes were visualized
with peroxidase chromogenic substrate (Kirkegaard & Perry
Laboratories Inc.). Sucrose-gradient purified FIPV and wild-type
raccoon pox virus-Vero cell lysates were used as the positive and
negative controls, respectively. A typical immunoblot is shown in
FIG. 8.
7. Raccoon Poxvirus Titration
Serial tenfold dilutions of virus are prepared in MEM and
inoculated in replicates of five onto Vero cells (1.times.10.sup.4
cells per well) in a 96 well plate. Virus preparations may be
pretreated by dilution into an equal volume of 0.5% trypsin and
incubation at 37.degree. C. for 30 min in order to release virus
from inclusions. Plates are incubated for 3-5 days at 37.degree. C.
(5% CO.sub.2) and observed for cytopathology typical of raccoon
poxvirus. Titers are calculated as 50% endpoints based on
cytopathology using the methods of Reed and Muench, The American
Journal of Hygiene 27(3):493-497) (1938).
EXAMPLE 2
PREPARATION OF VACCINE AND TESTING FOR EFFICACY IN CATS
1. Preparation of Master Seeds of RRPV-FIPV N and E1 Viruses
A single clone of each recombinant virus was selected for
large-scale expansion to serve as a master seed virus. The criteria
for selection were: 1) Demonstration of purity. Polymerase chain
reaction was utilized to insure that the clone was uncontaminated
with wild type virus. 2) Demonstration of adequate recombinant
proten expression by Western blot or other antigen detection
methods.
All recombinant virus expansions and titrations were done on Vero
cells in MEM containing 2.5% FBS. Each plaque purified virus clone
was expanded by inoculating a confluent monolayer of Vero cells in
a 150 cm.sup.2 flask (1.times.10.sup.7 cells) with 1 ml of
viral-cell lysate (approximately 10.sup.7 infectious virus
particles), and incubating at 37.degree. C. (5% CO.sub.2) until
100% cytopathic effect was observed (2-3 days). This virus-cell
lysate was titrated on Vero cells as described in Example 1, and
served as a premaster seed virus stock to obtain the master seed
virus. The MOI to be used to produce the highest titer master seed
virus was determined by inoculating a confluent monolayer of Vero
cells in a roller bottle (1.times.10.sup.8 cells) with various MOIs
of recombinant virus (e.g. 0.1, 0.05, 0.01, 0.005, and 0.001
TCID.sub.50 /cell.) The infected cells were incubated at 37.degree.
C. until greater than 80% CPE was observed (approximately 3 days),
and the titers of each infected roller bottle was determined. The
master seed viruses were aliquoted into 1.5 ml ampules, which were
sealed and stored in a liquid nitrogen freezer.
2. Preparation of Vaccines
3.times.10.sup.7 Vero cells were seeded into 850 cm.sup.2 roller
bottles in 200 ml of growth media (MEM containing 0.5% lactalbumin
hydrolysate and 5% FBS) and incubated for 18 hours at 37.degree. C.
The next day, the medium was removed from the cells and replaced
with 50ml of RRPV-FIPV N virus diluted to an MOI of 0.01 in
infection media (MEM containing 0.5% lactalbumin hydrolysate and
2.5% FBS). The virus used was at the second passage beyond the
master seed preparation. Virus was allowed to absorb to the cells
for 30 min at 37.degree. C., after which the volume of medium was
adjusted to 150 ml per roller bottle. Roller bottles were incubated
at 37.degree. C. until 100% cytopathology was evident (3 days). The
virus-cell lysate was harvested and stored frozen (-70.degree. C.).
The virus titer was determined to be 10.sup.6.97 TCID.sub. /ml.
RRPV-FIPV E1 stocks were prepared in the same manner, except that
an MOI of 0.1 was used. The final virus preparation was titered and
found to contain 10.sup.6.5 TCID.sub.50 /ml. Wild type raccoon
poxvirus was grown using the same methods as described above, and
contained 10.sup.6.44 TCID.sub.50 /ml.
3. Vaccination
A group of twenty-four 9-month-old cats (specific pathogen-free,
Harlan Sprague Dawley, Madison, Wis.), comprising seven males and
seventeen females, was used to demonstrate the efficacy of the
RRPV-FIPV N vaccine. Cats were divided into five groups and
vaccinated twice, 21 days apart, as indicated below:
______________________________________ # Volume Viral Dose
Vaccinaton Group Cats Vaccine (ml) (TCID.sub.50) Route*
______________________________________ 1 5 RRPV-FIPV N 3
10.sup.7.44 SC 2 5 RRPV-FIPV N 1 10.sup.6.97 IM 3 5 RRPV-FIPV N 3
10.sup.7.44 ORAL 4 4 RRPV-FIPV N 3 10.sup.6.44 SC (1:10 Dilution) 5
5 Wild Type REV 3 10.sup.6.44 SC
______________________________________ *SC = Subcutaneous IM =
Intramuscular Oral = Oral
4. Challenge
Two weeks following the second vaccination, cats were orally
inoculated with 10.sup.3.4 TCID.sub.50 of Feline Enteric
Coronavirus (strain 79-1683, ATCC VR-989). This virus induces a
subclinical infection which can enhance subsequent FIPV infection.
Three weeks later, cats were orally challenged with 10.sup.3.4
TCID.sub.50 of FIPV (strain 79-1146, ATCC VR-990). Cats were
monitored weekly for a total of 64 days after challenge for signs
of clinical disease including: fever, icterus, leukopenia, anemia,
weight loss, anorexia, depression, dehydration, and peritoneal
swelling. Cats deemed moribund were euthanized by the attending
veterinarian and post-mortem pathological examination was
performed. Clinical disease signs were scored as follows:
______________________________________ SIGN SCORE
______________________________________ Fever 103.0-10.39.degree. F.
1 point/day* 104.0-104.9.degree. F. 2 points/day
.gtoreq.105.0.degree. F. 3 points/day Dehydration 1 point/day
Depression 1 point/day Anorexia 1 point/day Peritoneal Swelling 1
point/day Icterus 1 point/day Weight Loss >20% 1 point per
observation >30% 2 points per observation >50% 5 points per
observation Leukopenia decrease of 50% 3 points per observation
counts <6000 2 points per observation Hematocrit 3 points per
observation <25% PCV Death 25 points
______________________________________ *For cats with baseline
temperatures averaging 103.degree. F., no points will be scored
until temperatures are in excess of 1.degree. F. above
baseline.
5. Evaluation of Induced Immunity to FIPV
Inoculation with virulent FIPV induced a fatal infection in 4/5
(80%) of the control cats, which were vaccinated with wild type
raccoon poxvirus (Table 1). Both effusive and non-effusive forms of
the disease were noted in the control cats. On the other hand,
clinical disease was essentially absent after challenge of the
subcutaneous vaccinates. The sporadic fever in these cats could be
attributed to excitability and the slight anemia on one day in cat
1264 is not a significant finding. The subcutaneous vaccinates
showed a statistically significant reduction in clinical signs
(p<0.05, by ANOVA) and death (p<0.01, by Chi Square Analysis)
when compared to the control cats.
The intramuscular route of vaccination was less effective in that
2/5 (40%) of the cats succumbed to FIPV-induced disease. However,
the onset of disease in these cats was delayed when compared to the
controls. The decreased efficacy may be related to the lower titer
of virus inoculated into these cats because only a 1 ml dose could
be administered by this route. There was also decreased efficacy
when cats were inoculated by the oral route (60% mortality) which
may indicate the need for a higher virus dose when vaccinated by
this route.
The protection conferred against FIPV-caused disease by the
subcutaneously administered vaccine was shown to be dose-dependent,
confirming the benefit of a high-titer RRPV-FIPV vaccine in
inducing protection against clinical disease induced by FIPV virus.
A suitable vaccine dose contains viral antigen in the range of
10.sup.4 -10.sup.8 TCID.sub.50 /ml, preferably 10.sup.7 -10.sup.8
TCID.sub.50 /ml. A typical dose for administration to cats is 1-3
ml, and delivery by the subcutaneous route is preferred.
TABLE 1
__________________________________________________________________________
TOTAL CLINICAL SCORES FOLLOWING CHALLENGE WITH FIPV CAT ID Fever
Weight Loss Leukopenia Anemia Chemical Signs Death* Total Score
__________________________________________________________________________
SUBCUTANEOUS VACCINATES 1260 0 0 0 0 0 0 0 1262 1 0 0 0 0 0 1 1264
1 0 0 0 0 0 4 1266 0 0 0 0 0 0 0 1268 2 0 0 0 0 0 2 INTRAMUSCULUAR
VACCINATES 1270 13 6 0 0 33 25 77 1272 2 0 0 0 0 0 2 1297 23 0 0 0
34 25 91 1301 3 0 0 0 0 0 3 ORAL VACCINATES 1303 1 0 4 0 0 0 5 1305
0 1 0 0 5 0 6 1307 3 1 0 0 4 25 33 1309 2 1 6 0 6 25 40 1311 4 3 0
0 18 25 50 1/10 DOSE SUBCUTANEOUS VACCINATES 1313 29 5 9 3 36 25
107 1315 17 0 0 0 20 25 62 1317 3 0 3 0 9 25 40 1319 1 0 0 0 6 0 7
CONTROLS 1321 39 3 18 3 63 25 151 1323 5 0 2 0 14 25 46 1327 0 1 2
0 15 25 43 1329 5 1 6 0 11 25 48 1337 1 0 0 0 0 0 1
__________________________________________________________________________
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 10 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 789 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline infectious
peritonitis virus (vii) IMMEDIATE SOURCE: (B) CLONE: FIPV E1 (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATGAAGTACATTTTGCTAATACTCGCGTGCATAATTGCATGCGTTTATGGTGAACGCTAC60
TGTGCCATGCAAGACAGTGGCTTGCAGTGTATTAATGGCACAAATTCAAGATGTCAAACC120
TGCTTTGAACGTGGTGATCTTATTTGGCATCTTGCTAACTGGAACTTCAGCTGGTCTGTA180
ATATTGATTGTTTTTATAACAGTGTTACAATATGGCAGACCACAATTTAGCTGGCTCGTT240
TATGGCATTAAAATGCTGATCATGTGGCTATTATGGCCTATTGTTCTAGCGCTTACGATT300
TTTAATGCATACTCTGAGTACCAAGTTTCCAGATATGTAATGTTCGGCTTTAGTGTTGCA360
GGTGCAGTTGTAACGTTTGCACTTTGGATGATGTATTTTGTGAGATCTGTTCAGCTATAT420
AGAAGAACCAAATCATGGTGGTCTTTTAATCCTGAGACTAATGCAATTCTTTGTGTTAAT480
GCATTGGGTAGAAGTTATGTGCTTCCCTTAGATGGTACTCCTACAGGTGTTACCCTTACT540
CTACTTTCAGGAAATCTATATGCTGAAGGTTTCAAAATGGCTGGTGGTTTAACCATCGAG600
CATTTGCCTAAATACGTCATGATTGCTACACCTAGTAGAACCATCGTTTATACATTAGTT660
GGAAAACAATTAAAAGCAACTACTGCCACAGGATGGGCTTACTACGTAAAATCTAAAGCT720
GGTGATTACTCAACAGAAGCACGTACTGACAATTTGAGTGAACATGAAAAATTATTACAT780
ATGGTGTAA789 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 1134 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline infectious
peritonitis virus (vii) IMMEDIATE SOURCE: (B) CLONE: FIPV N (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:2:
ATGGCCACACAGGGACAACGCGTCAACTGGGGAGATGAACCTTCCAAAAGACGTGGTCGT60
TCTAACTCTCGTGGTCGGAAGAATAATGATATACCTTTGTCATTCTACAACCCCATTACC120
CTCGAACAAGGATCTAAATTTTGGAATTTATGTCCGAGAGACCTTGTTCCCAAAGGAATA180
GGTAATAAGGATCAACAAATTGGTTATTGGAATAGACAGATTCGTTATCGTATTGTAAAA240
GGCCAGCGTAAGGAACTCGCTGAGAGGTGGTTCTTTTACTTCTTAGGTACAGGACCTCAT300
GCTGATGCTAAATTCAAAGACAAGATTGATGGAGTCTTCTGGGTTGCAAGGGATGGTGCC360
ATGAACAAGCCCACAACGCTTGGCACTCGTGGAACCAATAACGAATCCAAACCACTGAGA420
TTTGATGGTAAGATACCGCCACAGTTTCAGCTTGAAGTGAACCGTTCTAGGAACAATTCA480
AGGTCTGGTTCTCAGTCTAGATCTGTTTCAAGAAACAGATCTCAATCTAGAGGAAGACAC540
CATTCCAATAACCAGAATAATAATGTTGAGGATACAATTGTAGCCGTGCTTGAAAAATTA600
GGTGTTACTGACAAACAAAGGTCACGTTCTAAACCTAGAGAACGTAGTGATTCCAAACCT660
AGGGACACAACACCTAAGAATGCCAACAAACACACCTGGAAGAAAACTGCAGGCAAGGGA720
GATGTGACAACTTTCTATGGTGCTAGAAGTAGTTCAGCTAACTTTGGTGATAGTGATCTC780
GTTGCCAATGGTAACGCTGCCAAATGCTACCCTCAGATAGCTGAATGTGTTCCATCAGTG840
TCTAGCATAATCTTTGGCAGTCAATGGTCTGCTGAAGAAGCTGGTGATCAAGTGAAAGTC900
ACGCTCACTCACACCTACTACCTGCCAAAGGATGATGCCAAAACTAGTCAATTCCTAGAA960
CAGATTGACGCTTACAAGCGACCTTCTGAAGTGGCTAAGGATCAGAGGCAAAGAAGATCC1020
CGTTCTAAGTCTGCTGATAAGAAGCCTGAGGAGTTGTCTGTAACTCTTGTGGAGGCATAC1080
ACAGATGTGTTTGATGACACACAGGTTGAGATGATTGATGAGGTTACGAACTAA1134 (2)
INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 8710 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE: (A) ORGANISM: Feline infectious peritonitis
virus (vii) IMMEDIATE SOURCE: (B) CLONE: psc11f1 (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:3:
CGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCT60
TAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTC120
TAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAA180
TATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTT240
GCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCT300
GAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATC360
CTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTA420
TGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACAC480
TATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGC540
ATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAAC600
TTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGG660
GATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGAC720
GAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGC780
GAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTT840
GCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGA900
GCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCC960
CGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAG1020
ATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCA1080
TATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATC1140
CTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCA1200
GACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGC1260
TGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA1320
CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTT1380
CTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTC1440
GCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGG1500
TTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCG1560
TGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAG1620
CATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGC1680
AGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTAT1740
AGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGG1800
GGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGC1860
TGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATT1920
ACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCA1980
GTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCG2040
ATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAAC2100
GCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCG2160
GCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGAC2220
CATGATTACGCCAAGCTTTTGCGATCAATAAATGGATCACAACCAGTATCTCTTAACGAT2280
GTTCTTCGCAGATGATGATTCATTTTTTAAGTATTTGGCTAGTCAAGATGATGAAATCTT2340
CATTATCTGATATATTGCAAATCACTCAATATCTAGACTTTCTGTTATTATTATTGATCC2400
AATCAAAAAATAAATTAGAAGCCGTGGGTCATTGTTATGAATCTCTTTCAGAGGAATACA2460
GACAATTGACAAAATTCACAGACTTTCAAGATTTTAAAAAACTGTTTAACAAGGTCCCTA2520
TTGTTACAGATGGAAGGGTCAAACTTAATAAAGGATATTTGTTCGACTTTGTGATTAGTT2580
TGATGCGATTCAAAAAAGAATCCTCTCTAGCTACCACCGCAATAGATCCTGTTAGATACA2640
TAGATCCTCGTCGCAATATCGCATTTTCTAACGTGATGGATATATTAAAGTCGAATAAAG2700
TGAACAATAATTAATTCTTTATTGTCATCATGAACGGCGGACATATTCAGTTGATAATCG2760
GCCCCATGTTTTCAGGTAAAAGTACAGAATTAATTAGACGAGTTAGACGTTATCAAATAG2820
CTCAATATAAATGCGTGACTATAAAATATTCTAACGATAATAGATACGGAACGGGACTAT2880
GGACGCATGATAAGAATAATTTTGAAGCATTGGAAGCAACTAAACTATGTGATCTCTTGG2940
AATCAATTACAGATTTCTCCGTGATAGGTATCGATGAAGGACAGTTCTTTCCAGACATTG3000
TTGAATTCCGAGCTTGGCTGCAGGTCGGGGATCCCCCCTGCCCGGTTATTATTATTTTTG3060
ACACCAGACCAACTGGTAATGGTAGCGAACGGCGCTCAGCTGAATTCCGCCGATACTGAC3120
GGGCTCCAGGAGTCGTCGCCACCAATCCCCATATGGAAACCGTCGATATTCAGCCATGTG3180
CCTTCTTCCGCGTGCAGCAGATGGCGATGGCTGGTTTCCATCAGTTGCTGTTGACTGTAG3240
CGGCTGATGTTGAACTGGAAGTCGCCGCGCCACTGGTGTGGGCCATAATTCAATTCGCGC3300
GTCCCGCAGCGCAGACCGTTTTCGCTCGGGAAGACGTACGGGGTATACATGTCTGACAAT3360
GGCAGATCCCAGCGGTCAAAACAGGCGGCAGTAAGGCGGTCGGGATAGTTTTCTTGCGGC3420
CCTAATCCGAGCCAGTTTACCCGCTCTGCTACCTGCGCCAGCTGGCAGTTCAGGCCAATC3480
CGCGCCGGATGCGGTGTATCGCTCGCCACTTCAACATCAACGGTAATCGCCATTTGACCA3540
CTACCATCAATCCGGTAGGTTTTCCGGCTGATAAATAAGGTTTTCCCCTGATGCTGCCAC3600
GCGTGACCGGTCGTAATCAGCACCGCATCAGCAAGTGTATCTGCCGTGCACTGCAACAAC3660
GCTGCTTCGGCCTGGTAATGGCCCGCCGCCTTCCAGCGTTCGACCCAGGCGTTAGGGTCA3720
ATGCGGGTCGCTTCACTTACGCCAATGTCGTTATCCAGCGGTGCACGGGTGAACTGATCG3780
CGCAGCGGCGTCAGCAGTTGTTTTTTATCGCCAATCCACATCTGTGAAAGAAAGCCTGAC3840
TGGCGGTTAAATTGCCAACGCTTATTACCCAGCTCGATGCAAAAATCCATTTCGCTGGTG3900
GTCAGATGCGGGATGGCGTGGGACGCGGCGGGGAGCGTCACACTGAGGTTTTCCGCCAGA3960
CGCCACTGCTGCCAGGCGCTGATGTGCCCGGCTTCTGACCATGCGGTCGCGTTCGGTTGC4020
ACTACGCGTACTGTGAGCCAGAGTTGCCCGGCGCTCTCCGGCTGCGGTAGTTCAGGCAGT4080
TCAATCAACTGTTTACCTTGTGGAGCGACATCCAGAGGCACTTCACCGCTTGCCAGCGGC4140
TTACCATCCAGCGCCACCATCCAGTGCAGGAGCTCGTTATCGCTATGACGGAACAGGTAT4200
TCGCTGGTCACTTCGATGGTTTGCCCGGATAAACGGAACTGGAAAAACTGCTGCTGGTGT4260
TTTGCTTCCGTCAGCGCTGGATGCGGCGTGCGGTCGGCAAAGACCAGACCGTTCATACAG4320
AACTGGCGATCGTTCGGCGTATCGCCAAAATCACCGCCGTAAGCCGACCACGGGTTGCCG4380
TTTTCATCATATTTAATCAGCGACTGATCCACCCAGTCCCAGACGAAGCCGCCCTGTAAA4440
CGGGGATACTGACGAAACGCCTGCCAGTATTTAGCGAAACCGCCAAGACTGTTACCCATC4500
GCGTGGGCGTATTCGCAAAGGATCAGCGGGCGCGTCTCTCCAGGTAGCGAAAGCCATTTT4560
TTGATGGACCATTTCGGCACAGCCGGGAAGGGCTGGTCTTCATCCACGCGCGCGTACATC4620
GGGCAAATAATATCGGTGGCCGTGGTGTCGGCTCCGCCGCCTTCATACTGCACCGGGCGG4680
GAAGGATCGACAGATTTGATCCAGCGATACAGCGCGTCGTGATTAGCGCCGTGGCCTGAT4740
TCATTCCCCAGCGACCAGATGATCACACTCGGGTGATTACGATCGCGCTGCACCATTCGC4800
GTTACGCGTTCGCTCATCGCCGGTAGCCAGCGCGGATCATCGGTCAGACGATTGATTGGC4860
ACCATGCCGTGGGTTTCAATATTGGCTTCATCCACCACATACAGGCCGTAGCGGTCGCAC4920
AGCGTGTACCACAGCGGATGGTTCGGATAATGCGAACAGCGCACGGCGTTAAAGTTGTTC4980
TGCTTCATCAGCAGGATATCCTGCACCATCGTCTGCTCATCCATGACCTGACCATGCAGA5040
GGATGATGCTCGTGACGGTTAACGCCTCGAATCAGCAACGGCTTGCCGTTCAGCAGCAGC5100
AGACCATTTTCAATCCGCACCTCGCGGAAACCGACATCGCAGGCTTCTGCTTCAATCAGC5160
GTGCCGTCGGCGGTGTGCAGTTCAACCACCGCACGATAGAGATTCGGGATTTCGGCGCTC5220
CACAGTTTCGGGTTTTCGACCTTGAGACGTAGTGTGACGCGATCGGCATAACCACCACGC5280
TCATCGATAATTTCACCGCCGAAAGGCGCGGTGCCGCTGGCGACCTGCGTTTCACCCTGC5340
CATAAAGAAACTGTTACCCGTAGGTAGTCACGCAACTCGCCGCACATCTGAACTTCAGCC5400
TCCAGTACAGCGCGGCTGAAATCATCATTAAAGCGAGTGGCAACATGGAAATCGCTGATT5460
TGTGTAGTCGGTTTATGCAGCAACGAGACGTCACGGAAAATGCCGCTCATCCGCCACATA5520
TCCTGATCTTCCAGATAACTGCCGTCACTCCAACGCAGCACCATCACCGCGAGGCGGTTT5580
TCTCCGGCGCGTAAAAATGCGCTCAGGTCAAATTCAGACGGCAAACGACTGTCCTGGCCG5640
TAACCGACCCAGCGCCCGTTGCACCACAGATGAAACGCCGAGTTAACGCCATCAAAAATA5700
ATTCGCGTCTGGCCTTCCTGTAGCCAGCTTTCATCAACATTAAATGTGAGCGAGTAACAA5760
CCCGTCGGATTCTCCGTGGGAACAAACGGCGGATTGACCGTAATGGGATAGGTTACGTTG5820
GTGTAGATGGGCGCATCGTAACCGTGCATCTGCCAGTTTGAGGGGACGACGACAGTATCG5880
GCCTCAGGAAGATCGCACTCCAGCCAGCTTTCCGGCACCGCTTCTGGTGCCGGAAACCAG5940
GCAAAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCC6000
TCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTA6060
ACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGGATCCCTCGAGGAATTCATT6120
TATAGCATAGAAAAAAACAAAATGAAATTCTACTATATTTTTACATACATATATTCTAAA6180
TATGAAAGTGGTGATTGTGACTAGCGTAGCATCGCTTCTAGACATATACTATATAGTAAT6240
ACCAATACTCAAGACTACGAAACTGATACAATCTCTTATCATGTGGGTAATGTTCTCGAT6300
GTCGAATAGCCATATGCCGGTAGTTGCGATATACATAAACTGATCACTAATTCCAAACCC6360
ACCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAATTTCTCG6420
TAAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGAATCATAAAGAACA6480
GTGACGGATCCCAATTCGGGCATTTTTGGTTTGAACTAAACAAAATGAAGTACATTTTGC6540
TAATACTCGCGTGCATAATTGCATGCGTTTATGGTGAACGCTACTGTGCCATGCAAGACA6600
GTGGCTTGCAGTGTATTAATGGCACAAATTCAAGATGTCAAACCTGCTTTGAACGTGGTG6660
ATCTTATTTGGCATCTTGCTAACTGGAACTTCAGCTGGTCTGTAATATTGATTGTTTTTA6720
TAACAGTGTTACAATATGGCAGACCACAATTTAGCTGGCTCGTTTATGGCATTAAAATGC6780
TGATCATGTGGCTATTATGGCCTATTGTTCTAGCGCTTACGATTTTTAATGCATACTCTG6840
AGTACCAAGTTTCCAGATATGTAATGTTCGGCTTTAGTGTTGCAGGTGCAGTTGTAACGT6900
TTGCACTTTGGATGATGTATTTTGTGAGATCTGTTCAGCTATATAGAAGAACCAAATCAT6960
GGTGGTCTTTTAATCCTGAGACTAATGCAATTCTTTGTGTTAATGCATTGGGTAGAAGTT7020
ATGTGCTTCCCTTAGATGGTACTCCTACAGGTGTTACCCTTACTCTACTTTCAGGAAATC7080
TATATGCTGAAGGTTTCAAAATGGCTGGTGGTTTAACCATCGAGCATTTGCCTAAATACG7140
TCATGATTGCTACACCTAGTAGAACCATCGTTTATACATTAGTTGGAAAACAATTAAAAG7200
CAACTACTGCCACAGGATGGGCTTACTACGTAAAATCTAAAGCTGGTGATTACTCAACAG7260
AAGCACGTACTGACAATTTGAGTGAACATGAAAAATTATTACATATGGTGTAACTAAACT7320
TTCAAATGGGGGAATTCTGTGAGCGTATGGCAAACGAAGGAAAAATTAGTTATAGTAGCC7380
GCACTCGATGGGACATTTCAACGTAAACCGTTTAATAATATTTTGAATCTTATTCCATTA7440
TCTGAAATGGTGGTAAAACTAACTGCTGTGTGTATGAAATGCTTTAAGGAGGCTTCCTTT7500
TCTAAACGATTGGGTGAGGAAACCGAGATAGAAATAATAGGAGGTAATGATATGTATCAA7560
TCGGTGTGTAGAAAGTGTTACATCGACTCATAATATTATATTTTTTATCTAAAAAACTAA7620
AAATAAACATTGATTAAATTTTAATATAATACTTAAAAATGGATGTTGTGTCGTTAGATA7680
AACCGTTTATGTATTTTGAGGAAATTGATAATGAGTTAGATTACGAACCAGAAAGTGCAA7740
ATGAGGTCGCAAAAAAACTGCCGTATCAAGGACAGTTAAAACTATTACTAGGAGAATTAT7800
TTTTTCTTAGTAAGTTACAGCGACACGGTATATTAGATGGTGCCACCGTAGTGTATATAG7860
GATCTGCTCCCGGTACACATATACGTTATTTGAGAGATCATTTCTATAATTTAGGAGTGA7920
TCATCAAATGGATGCTAATTGACGGCCGCCATCATGATCCTATTTTAAATGGATTGCGTG7980
ATGTGACTCTAGTGACTCGGTTCGTTGATGAGGAATATCTACGATCCATCAAAAAACAAC8040
TGCATCCTTCTAAGATTATTTTAATTTCTGATGTGAGATCCAAACGAGGAGGAAATGAAC8100
CTAGTACGGCGGATTTACTAAGTAATTACGCTCTACAAAATGTCATGATTAGTATTTTAA8160
ACCCCGTGGCGTCTAGTCTTAAATGGAGATGCCCGTTTCCAGATCAATGGATCAAGGACT8220
TTTATATCCCACACGGTAATAAAATGTTACAACCTTTTGCTCCTTCATATTCAGGGCCGT8280
CGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGC8340
ACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCA8400
ACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCTTTACGCATCT8460
GTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACCATCTGCTCTGATGCCGCATA8520
GTTAAGCCAGTACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCC8580
GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACA8640
AGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACG8700
CGCGAGGCAG8710 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 9019 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE:
DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Feline
immunodeficiency virus (vii) IMMEDIATE SOURCE: (B) CLONE: psc11e1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTT60
AGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCT120
AAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAAT180
ATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTG240
CGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTG300
AAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCC360
TTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTAT420
GTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACT480
ATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCA540
TGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACT600
TACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGG660
ATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACG720
AGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCG780
AACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTG840
CAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAG900
CCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCC960
GTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGA1020
TCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCAT1080
ATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCC1140
TTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAG1200
ACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT1260
GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC1320
CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTC1380
TAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCG1440
CTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGT1500
TGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGT1560
GCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC1620
ATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCA1680
GGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATA1740
GTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGG1800
GGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCT1860
GGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA1920
CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAG1980
TGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGA2040
TTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACG2100
CAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGG2160
CTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACC2220
ATGATTACGCCAAGCTTTTGCGATCAATAAATGGATCACAACCAGTATCTCTTAACGATG2280
TTCTTCGCAGATGATGATTCATTTTTTAAGTATTTGGCTAGTCAAGATGATGAAATCTTC2340
ATTATCTGATATATTGCAAATCACTCAATATCTAGACTTTCTGTTATTATTATTGATCCA2400
ATCAAAAAATAAATTAGAAGCCGTGGGTCATTGTTATGAATCTCTTTCAGAGGAATACAG2460
ACAATTGACAAAATTCACAGACTTTCAAGATTTTAAAAAACTGTTTAACAAGGTCCCTAT2520
TGTTACAGATGGAAGGGTCAAACTTAATAAAGGATATTTGTTCGACTTTGTGATTAGTTT2580
GATGCGATTCAAAAAAGAATCCTCTCTAGCTACCACCGCAATAGATCCTGTTAGATACAT2640
AGATCCTCGTCGCAATATCGCATTTTCTAACGTGATGGATATATTAAAGTCGAATAAAGT2700
GAACAATAATTAATTCTTTATTGTCATCATGAACGGCGGACATATTCAGTTGATAATCGG2760
CCCCATGTTTTCAGGTAAAAGTACAGAATTAATTAGACGAGTTAGACGTTATCAAATAGC2820
TCAATATAAATGCGTGACTATAAAATATTCTAACGATAATAGATACGGAACGGGACTATG2880
GACGCATGATAAGAATAATTTTGAAGCATTGGAAGCAACTAAACTATGTGATCTCTTGGA2940
ATCAATTACAGATTTCTCCGTGATAGGTATCGATGAAGGACAGTTCTTTCCAGACATTGT3000
TGAATTCCGAGCTTGGCTGCAGGTCGGGGATCCCCCCTGCCCGGTTATTATTATTTTTGA3060
CACCAGACCAACTGGTAATGGTAGCGAACGGCGCTCAGCTGAATTCCGCCGATACTGACG3120
GGCTCCAGGAGTCGTCGCCACCAATCCCCATATGGAAACCGTCGATATTCAGCCATGTGC3180
CTTCTTCCGCGTGCAGCAGATGGCGATGGCTGGTTTCCATCAGTTGCTGTTGACTGTAGC3240
GGCTGATGTTGAACTGGAAGTCGCCGCGCCACTGGTGTGGGCCATAATTCAATTCGCGCG3300
TCCCGCAGCGCAGACCGTTTTCGCTCGGGAAGACGTACGGGGTATACATGTCTGACAATG3360
GCAGATCCCAGCGGTCAAAACAGGCGGCAGTAAGGCGGTCGGGATAGTTTTCTTGCGGCC3420
CTAATCCGAGCCAGTTTACCCGCTCTGCTACCTGCGCCAGCTGGCAGTTCAGGCCAATCC3480
GCGCCGGATGCGGTGTATCGCTCGCCACTTCAACATCAACGGTAATCGCCATTTGACCAC3540
TACCATCAATCCGGTAGGTTTTCCGGCTGATAAATAAGGTTTTCCCCTGATGCTGCCACG3600
CGTGACCGGTCGTAATCAGCACCGCATCAGCAAGTGTATCTGCCGTGCACTGCAACAACG3660
CTGCTTCGGCCTGGTAATGGCCCGCCGCCTTCCAGCGTTCGACCCAGGCGTTAGGGTCAA3720
TGCGGGTCGCTTCACTTACGCCAATGTCGTTATCCAGCGGTGCACGGGTGAACTGATCGC3780
GCAGCGGCGTCAGCAGTTGTTTTTTATCGCCAATCCACATCTGTGAAAGAAAGCCTGACT3840
GGCGGTTAAATTGCCAACGCTTATTACCCAGCTCGATGCAAAAATCCATTTCGCTGGTGG3900
TCAGATGCGGGATGGCGTGGGACGCGGCGGGGAGCGTCACACTGAGGTTTTCCGCCAGAC3960
GCCACTGCTGCCAGGCGCTGATGTGCCCGGCTTCTGACCATGCGGTCGCGTTCGGTTGCA4020
CTACGCGTACTGTGAGCCAGAGTTGCCCGGCGCTCTCCGGCTGCGGTAGTTCAGGCAGTT4080
CAATCAACTGTTTACCTTGTGGAGCGACATCCAGAGGCACTTCACCGCTTGCCAGCGGCT4140
TACCATCCAGCGCCACCATCCAGTGCAGGAGCTCGTTATCGCTATGACGGAACAGGTATT4200
CGCTGGTCACTTCGATGGTTTGCCCGGATAAACGGAACTGGAAAAACTGCTGCTGGTGTT4260
TTGCTTCCGTCAGCGCTGGATGCGGCGTGCGGTCGGCAAAGACCAGACCGTTCATACAGA4320
ACTGGCGATCGTTCGGCGTATCGCCAAAATCACCGCCGTAAGCCGACCACGGGTTGCCGT4380
TTTCATCATATTTAATCAGCGACTGATCCACCCAGTCCCAGACGAAGCCGCCCTGTAAAC4440
GGGGATACTGACGAAACGCCTGCCAGTATTTAGCGAAACCGCCAAGACTGTTACCCATCG4500
CGTGGGCGTATTCGCAAAGGATCAGCGGGCGCGTCTCTCCAGGTAGCGAAAGCCATTTTT4560
TGATGGACCATTTCGGCACAGCCGGGAAGGGCTGGTCTTCATCCACGCGCGCGTACATCG4620
GGCAAATAATATCGGTGGCCGTGGTGTCGGCTCCGCCGCCTTCATACTGCACCGGGCGGG4680
AAGGATCGACAGATTTGATCCAGCGATACAGCGCGTCGTGATTAGCGCCGTGGCCTGATT4740
CATTCCCCAGCGACCAGATGATCACACTCGGGTGATTACGATCGCGCTGCACCATTCGCG4800
TTACGCGTTCGCTCATCGCCGGTAGCCAGCGCGGATCATCGGTCAGACGATTGATTGGCA4860
CCATGCCGTGGGTTTCAATATTGGCTTCATCCACCACATACAGGCCGTAGCGGTCGCACA4920
GCGTGTACCACAGCGGATGGTTCGGATAATGCGAACAGCGCACGGCGTTAAAGTTGTTCT4980
GCTTCATCAGCAGGATATCCTGCACCATCGTCTGCTCATCCATGACCTGACCATGCAGAG5040
GATGATGCTCGTGACGGTTAACGCCTCGAATCAGCAACGGCTTGCCGTTCAGCAGCAGCA5100
GACCATTTTCAATCCGCACCTCGCGGAAACCGACATCGCAGGCTTCTGCTTCAATCAGCG5160
TGCCGTCGGCGGTGTGCAGTTCAACCACCGCACGATAGAGATTCGGGATTTCGGCGCTCC5220
ACAGTTTCGGGTTTTCGACCTTGAGACGTAGTGTGACGCGATCGGCATAACCACCACGCT5280
CATCGATAATTTCACCGCCGAAAGGCGCGGTGCCGCTGGCGACCTGCGTTTCACCCTGCC5340
ATAAAGAAACTGTTACCCGTAGGTAGTCACGCAACTCGCCGCACATCTGAACTTCAGCCT5400
CCAGTACAGCGCGGCTGAAATCATCATTAAAGCGAGTGGCAACATGGAAATCGCTGATTT5460
GTGTAGTCGGTTTATGCAGCAACGAGACGTCACGGAAAATGCCGCTCATCCGCCACATAT5520
CCTGATCTTCCAGATAACTGCCGTCACTCCAACGCAGCACCATCACCGCGAGGCGGTTTT5580
CTCCGGCGCGTAAAAATGCGCTCAGGTCAAATTCAGACGGCAAACGACTGTCCTGGCCGT5640
AACCGACCCAGCGCCCGTTGCACCACAGATGAAACGCCGAGTTAACGCCATCAAAAATAA5700
TTCGCGTCTGGCCTTCCTGTAGCCAGCTTTCATCAACATTAAATGTGAGCGAGTAACAAC5760
CCGTCGGATTCTCCGTGGGAACAAACGGCGGATTGACCGTAATGGGATAGGTTACGTTGG5820
TGTAGATGGGCGCATCGTAACCGTGCATCTGCCAGTTTGAGGGGACGACGACAGTATCGG5880
CCTCAGGAAGATCGCACTCCAGCCAGCTTTCCGGCACCGCTTCTGGTGCCGGAAACCAGG5940
CAAAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCT6000
CTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAA6060
CGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGGATCCCTCGAGGAATTCATTT6120
ATAGCATAGAAAAAAACAAAATGAAATTCTACTATATTTTTACATACATATATTCTAAAT6180
ATGAAAGTGGTGATTGTGACTAGCGTAGCATCGCTTCTAGACATATACTATATAGTAATA6240
CCAATACTCAAGACTACGAAACTGATACAATCTCTTATCATGTGGGTAATGTTCTCGATG6300
TCGAATAGCCATATGCCGGTAGTTGCGATATACATAAACTGATCACTAATTCCAAACCCA6360
CCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAATTTCTCGT6420
AAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGAATCATAAAGAACAG6480
TGACGGATCCCGGGATGGCCACACAGGGACAACGCGTCAACTGGGGAGATGAACCTTCCA6540
AAAGACGTGGTCGTTCTAACTCTCGTGGTCGGAAGAATAATGATATACCTTTGTCATTCT6600
ACAACCCCATTACCCTCGAACAAGGATCTAAATTTTGGAATTTATGTCCGAGAGACCTTG6660
TTCCCAAAGGAATAGGTAATAAGGATCAACAAATTGGTTATTGGAATAGACAGATTCGTT6720
ATCGTATTGTAAAAGGCCAGCGTAAGGAACTCGCTGAGAGGTGGTTCTTTTACTTCTTAG6780
GTACAGGACCTCATGCTGATGCTAAATTCAAAGACAAGATTGATGGAGTCTTCTGGGTTG6840
CAAGGGATGGTGCCATGAACAAGCCCACAACGCTTGGCACTCGTGGAACCAATAACGAAT6900
CCAAACCACTGAGATTTGATGGTAAGATACCGCCACAGTTTCAGCTTGAAGTGAACCGTT6960
CTAGGAACAATTCAAGGTCTGGTTCTCAGTCTAGATCTGTTTCAAGAAACAGATCTCAAT7020
CTAGAGGAAGACACCATTCCAATAACCAGAATAATAATGTTGAGGATACAATTGTAGCCG7080
TGCTTGAAAAATTAGGTGTTACTGACAAACAAAGGTCACGTTCTAAACCTAGAGAACGTA7140
GTGATTCCAAACCTAGGGACACAACACCTAAGAATGCCAACAAACACACCTGGAAGAAAA7200
CTGCAGGCAAGGGAGATGTGACAACTTTCTATGGTGCTAGAAGTAGTTCAGCTAACTTTG7260
GTGATAGTGATCTCGTTGCCAATGGTAACGCTGCCAAATGCTACCCTCAGATAGCTGAAT7320
GTGTTCCATCAGTGTCTAGCATAATCTTTGGCAGTCAATGGTCTGCTGAAGAAGCTGGTG7380
ATCAAGTGAAAGTCACGCTCACTCACACCTACTACCTGCCAAAGGATGATGCCAAAACTA7440
GTCAATTCCTAGAACAGATTGACGCTTACAAGCGACCTTCTGAAGTGGCTAAGGATCAGA7500
GGCAAAGAAGATCCCGTTCTAAGTCTGCTGATAAGAAGCCTGAGGAGTTGTCTGTAACTC7560
TTGTGGAGGCATACACAGATGTGTTTGATGACACACAGGTTGAGATGATTGATGAGGTTA7620
CGAACTAAACGCATGCCCGGGAATTCTGTGAGCGTATGGCAAACGAAGGAAAAATTAGTT7680
ATAGTAGCCGCACTCGATGGGACATTTCAACGTAAACCGTTTAATAATATTTTGAATCTT7740
ATTCCATTATCTGAAATGGTGGTAAAACTAACTGCTGTGTGTATGAAATGCTTTAAGGAG7800
GCTTCCTTTTCTAAACGATTGGGTGAGGAAACCGAGATAGAAATAATAGGAGGTAATGAT7860
ATGTATCAATCGGTGTGTAGAAAGTGTTACATCGACTCATAATATTATATTTTTTATCTA7920
AAAAACTAAAAATAAACATTGATTAAATTTTAATATAATACTTAAAAATGGATGTTGTGT7980
CGTTAGATAAACCGTTTATGTATTTTGAGGAAATTGATAATGAGTTAGATTACGAACCAG8040
AAAGTGCAAATGAGGTCGCAAAAAAACTGCCGTATCAAGGACAGTTAAAACTATTACTAG8100
GAGAATTATTTTTTCTTAGTAAGTTACAGCGACACGGTATATTAGATGGTGCCACCGTAG8160
TGTATATAGGATCTGCTCCCGGTACACATATACGTTATTTGAGAGATCATTTCTATAATT8220
TAGGAGTGATCATCAAATGGATGCTAATTGACGGCCGCCATCATGATCCTATTTTAAATG8280
GATTGCGTGATGTGACTCTAGTGACTCGGTTCGTTGATGAGGAATATCTACGATCCATCA8340
AAAAACAACTGCATCCTTCTAAGATTATTTTAATTTCTGATGTGAGATCCAAACGAGGAG8400
GAAATGAACCTAGTACGGCGGATTTACTAAGTAATTACGCTCTACAAAATGTCATGATTA8460
GTATTTTAAACCCCGTGGCGTCTAGTCTTAAATGGAGATGCCCGTTTCCAGATCAATGGA8520
TCAAGGACTTTTATATCCCACACGGTAATAAAATGTTACAACCTTTTGCTCCTTCATATT8580
CAGGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCG8640
CCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCG8700
CCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCTT8760
TACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACCATCTGCTCTGA8820
TGCCGCATAGTTAAGCCAGTACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCC8880
CCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGC8940
TTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATC9000
ACCGAAACGCGCGAGGCAG9019 (2) INFORMATION FOR SEQ ID NO:5: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 262 amino acids (B) TYPE:
amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM:
Feline infectious peritonitis virus (vii) IMMEDIATE SOURCE: (B)
CLONE: FIPV E1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
MetLysTyrIleLeuLeuIleLeuAlaCysIleIleAlaCysValTyr 151015
GlyGluArgTyrCysAlaMetGlnAspSerGlyLeuGlnCysIleAsn 202530
GlyThrAsnSerArgCysGlnThrCysPheGluArgGlyAspLeuIle 354045
TrpHisLeuAlaAsnTrpAsnPheSerTrpSerValIleLeuIleVal 505560
PheIleThrValLeuGlnTyrGlyArgProGlnPheSerTrpLeuVal 65707580
TyrGlyIleLysMetLeuIleMetTrpLeuLeuTrpProIleValLeu 859095
AlaLeuThrIlePheAsnAlaTyrSerGluTyrGlnValSerArgTyr 100105110
ValMetPheGlyPheSerValAlaGlyAlaValValThrPheAlaLeu 115120125
TrpMetMetTyrPheValArgSerValGlnLeuTyrArgArgThrLys 130135140
SerTrpTrpSerPheAsnProGluThrAsnAlaIleLeuCysValAsn 145150155160
AlaLeuGlyArgSerTyrValLeuProLeuAspGlyThrProThrGly 165170175
ValThrLeuThrLeuLeuSerGlyAsnLeuTyrAlaGluGlyPheLys 180185190
MetAlaGlyGlyLeuThrIleGluHisLeuProLysTyrValMetIle 195200205
AlaThrProSerArgThrIleValTyrThrLeuValGlyLysGlnLeu 210215220
LysAlaThrThrAlaThrGlyTrpAlaTyrTyrValLysSerLysAla 225230235240
GlyAspTyrSerThrGluAlaArgThrAspAsnLeuSerGluHisGlu 245250255
LysLeuLeuHisMetVal 260 (2) INFORMATION FOR SEQ ID NO:6: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 377 amino acids (B) TYPE:
amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM:
Feline infectious peritonitis virus (vii) IMMEDIATE SOURCE: (B)
CLONE: FIPV N (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
MetAlaThrGlnGlyGlnArgValAsnTrpGlyAspGluProSerLys 151015
ArgArgGlyArgSerAsnSerArgGlyArgLysAsnAsnAspIlePro 202530
LeuSerPheTyrAsnProIleThrLeuGluGlnGlySerLysPheTrp 354045
AsnLeuCysProArgAspLeuValProLysGlyIleGlyAsnLysAsp 505560
GlnGlnIleGlyTyrTrpAsnArgGlnIleArgTyrArgIleValLys 65707580
GlyGlnArgLysGluLeuAlaGluArgTrpPhePheTyrPheLeuGly 859095
ThrGlyProHisAlaAspAlaLysPheLysAspLysIleAspGlyVal 100105110
PheTrpValAlaArgAspGlyAlaMetAsnLysProThrThrLeuGly 115120125
ThrArgGlyThrAsnAsnGluSerLysProLeuArgPheAspGlyLys 130135140
IleProProGlnPheGlnLeuGluValAsnArgSerArgAsnAsnSer 145150155160
ArgSerGlySerGlnSerArgSerValSerArgAsnArgSerGlnSer 165170175
ArgGlyArgHisHisSerAsnAsnGlnAsnAsnAsnValGluAspThr 180185190
IleValAlaValLeuGluLysLeuGlyValThrAspLysGlnArgSer 195200205
ArgSerLysProArgGluArgSerAspSerLysProArgAspThrThr 210215220
ProLysAsnAlaAsnLysHisThrTrpLysLysThrAlaGlyLysGly 225230235240
AspValThrThrPheTyrGlyAlaArgSerSerSerAlaAsnPheGly 245250255
AspSerAspLeuValAlaAsnGlyAsnAlaAlaLysCysTyrProGln 260265270
IleAlaGluCysValProSerValSerSerIleIlePheGlySerGln 275280285
TrpSerAlaGluGluAlaGlyAspGlnValLysValThrLeuThrHis 290295300
ThrTyrTyrLeuProLysAspAspAlaLysThrSerGlnPheLeuGlu 305310315320
GlnIleAspAlaTyrLysArgProSerGluValAlaLysAspGlnArg 325330335
GlnArgArgSerArgSerLysSerAlaAspLysLysProGluGluLeu 340345350
SerValThrLeuValGluAlaTyrThrAspValPheAspAspThrGln 355360365
ValGluMetIleAspGluValThrAsn 370375 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: Other nucleic acid (vi) ORIGINAL SOURCE:
(A) ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE
SOURCE: (B) CLONE: N primer #1 (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:7: CTCGTGGTCGGAAGAATAATGATA24 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: Other nucleic acid (vi) ORIGINAL SOURCE: (A)
ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE
SOURCE: (B) CLONE: N primer #2 (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:8: AGCACCATAGAAAGTTGTCACATC24 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: Other nucleic acid (vi) ORIGINAL SOURCE: (A)
ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE
SOURCE: (B) CLONE: E1 primer #1 (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:9: TATGTAATGTTCGGCTTTAGTG22 (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: Other nucleic acid (vi) ORIGINAL SOURCE: (A)
ORGANISM: Feline infectious peritonitis virus (vii) IMMEDIATE
SOURCE: (B) CLONE: E1 primer #2 (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:10: GTGCTTCTGTTGAGTAATCACC22
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